The role of PCNA as a scaffold protein in cellular signaling is functionally conserved between yeast and humans

Abstract

Proliferating cell nuclear antigen ( PCNA ), a member of the highly conserved DNA sliding clamp family, is an essential protein for cellular processes including DNA replication and repair. A large number of proteins from higher eukaryotes contain one of two PCNA ‐interacting motifs: PCNA ‐interacting protein box ( PIP box) and AlkB homologue 2 PCNA ‐interacting motif ( APIM ). APIM has been shown to be especially important during cellular stress. PIP box is known to be functionally conserved in yeast, and here, we show that this is also the case for APIM . Several of the 84 APIM ‐containing yeast proteins are associated with cellular signaling as hub proteins, which are able to interact with a large number of other proteins. Cellular signaling is highly conserved throughout evolution, and we recently suggested a novel role for PCNA as a scaffold protein in cellular signaling in human cells. A cell‐penetrating peptide containing the APIM sequence increases the sensitivity toward the chemotherapeutic agent cisplatin in both yeast and human cells, and both yeast and human cells become hypersensitive when the Hog1/p38 MAPK pathway is blocked. These results suggest that the interactions between APIM ‐containing signaling proteins and PCNA during the DNA damage response is evolutionary conserved between yeast and mammals and that PCNA has a role in cellular signaling also in yeast.

PCNA has specific functions in regulation of metabolism in haematological cells

“…The affinity of APIM to PCNA is increased during cellular stress (Ciccia et al, 2012;Gilljam et al, 2009) in accordance with the ability of the APIM-peptide to increase the efficacy of multiple anti-cancer drugs, including genotoxic drugs and drugs targeting microtubules or kinases (Gederaas et al, 2014;Müller et al, 2013;Olaisen et al, 2018;Olaisen et al, 2015;Sogaard, Blindheim, et al, 2018;Sogaard, Moestue, et al, 2018;Sogaard et al, 2019). Further, data supports impairment of PCNA´s scaffold roles in DNA repair, DNA translesion synthesis and cellular signaling after APIM-peptide treatment (Gilljam et al, 2012;Olaisen et al, 2015;Raeder et al, 2018).…”

“…Many of the APIM-or PIP-containing proteins are not directly involved in processing of DNA, but have important roles in cellular signaling, e.g. in the PI3K/AKT and MAPK pathways (Olaisen et al, 2018;Olaisen, Müller, Nedal, & Otterlei, 2015). Multi-layered regulatory mechanisms are required to control the repertoire of up to 600 proteins (Olaisen et al, 2018) that may interact with PCNA through these two motifs at any given time.…”

“…in the PI3K/AKT and MAPK pathways (Olaisen et al, 2018;Olaisen, Müller, Nedal, & Otterlei, 2015). Multi-layered regulatory mechanisms are required to control the repertoire of up to 600 proteins (Olaisen et al, 2018) that may interact with PCNA through these two motifs at any given time. These regulatory mechanisms include affinity driven competition, at least partly mediated via posttranslational modifications (PTMs) of PCNA or PCNA binding proteins, and/or the context of the PCNA complexes (e.g.…”

“…Several lines of evidence support cytosolic or non-canonical roles for PCNA: I) Cytosolic PCNA is reported to regulate apoptosis by binding to procaspases in mature neutrophils (Witko-Sarsat et al, 2010), multiple myeloma (MM) cells (Müller et al, 2013), and neuroblastoma cells (Yin et al, 2015); II) Inhibition of the ability of APIM-containing proteins to interact with PCNA by treating cells with a cellpenetrating peptide containing the APIM sequence (APIM-peptide) strongly reduced the secretion of cytokines from human monocytes stimulated with Toll-like receptor ligands (Olaisen et al, 2015); III) Cytosolic PCNA in cancer cells inhibits activation of natural killer cells, thereby helping cancer cells to evade the immune system (Rosental et al, 2011); IV) Glycolytic enzymes associate with PCNA, and nuclear export of PCNA correlated with increased Warburg-effect in acute myeloid leukaemia (AML) cells (Naryzhny & Lee, 2010;Ohayon et al, 2016); V) Multiple proteins involved in PI3K/AKT and MAPK signaling were found in PCNA pull-downs (Olaisen et al, 2015), and targeting PCNA has been linked to downregulation of EGFR and PI3K/AKT pathways in bladder cancer cells and monocytes (Olaisen et al, 2015;Sogaard, Blindheim, et al, 2018); VI) Cytosolic PCNA participates in reactive oxygen species (ROS) regulation via interaction with a subunit of the NADPH oxidase complex, p47phox (Ohayon et al, 2019); VII) Combination treatments with APIM-peptide and receptor tyrosine kinase inhibitors against EGFR/HER2/VEGFR and cMET showed increased anticancer activity in absence of any DNA-damaging agent (Sogaard et al, 2019); and VIII) A functional conservation of PCNA as a scaffold protein in MAPK signaling has been shown between mammalians and yeast (Olaisen et al, 2018). These cytosolic scaffold functions of PCNA, as well as the verified APIM-PCNA interactions important in DNA damage repair and/or tolerance, are illustrated in Figure 1.…”

“…(Bacquin et al, 2013;Fattah et al, 2014;Gilljam et al, 2009;Gilljam, Müller, Liabakk, & Otterlei, 2012;Raeder et al, 2018;Seelinger & Otterlei, 2020). Cytosolic roles: Multiple cytosolic proteins involved in cellular signaling and metabolism, including glycolytic enzymes, contain APIM or PIP-box motifs (Ohayon et al, 2019;Olaisen et al, 2018;Olaisen et al, 2015), or are found in complex with PCNA (Naryzhny & Lee, 2010;Olaisen et al, 2015;Witko-Sarsat et al, 2010). Studies have shown that impairing PCNA´s scaffold function affects multiple signaling pathways including the PI3K/AKT and MAPK pathways and the cytokine production after toll receptor stimuli (Olaisen et al, 2018;Olaisen et al, 2015;Sogaard et al, 2019), regulation of apoptosis (Müller et al, 2013;Witko-Sarsat et al, 2010;Yin et al, 2015), cytotoxicity of NK cells (Rosental et al, 2011;Shemesh et al, 2018), and cellular defense against ROS (Ohayon et al, 2019).…”